The invention relates to the field of recombinant DNA technology, more in particular to the field of gene therapy. In particular the invention relates to gene therapy using materials derived from adenovirus, in particular human recombinant adenovirus. It especially relates to novel virus derived vectors and novel packaging cell lines for vectors based on adenoviruses.
Gene therapy is a recently developed concept for which a wide range of applications can be and have been envisaged. In gene therapy a molecule carrying genetic information is introduced into some or all cells of a host, as a result of which the genetic information is added to the host in a functional format. Included is the treatment of genetic disorders by providing the genetic information for supplementing a protein or other substance which is, through said genetic disorder, not present or at least present in insufficient amounts in the host, the treatment of tumors and (other) acquired disease such as (auto) immune diseases or infections, or other processes. The genetic information added may be a gene or a derivative of a gene, such as a cDNA, which encodes a protein. In this case the functional format means that the protein can be expressed by the machinery of the host cell. The genetic information can also be a sequence of nucleotides complementary to a sequence of nucleotides (be it DNA or RNA) present in the host cell. The functional format in this case is that the added DNA (nucleic acid) molecule or copies made thereof in situ are capable of base pairing with the complementary sequence present in the host cell.
Thus, there are basically three different approaches in gene therapy, one directed towards compensating a deficiency present in a (mammalian) host; the second directed towards the removal or elimination of unwanted substances (organisms or cells) and the third directed towards application of a recombinant vaccine (tumors or foreign microorganisms).
For the purpose of gene therapy, adenoviruses carrying deletions have been proposed as suitable vehicles. Adenoviruses are non-enveloped DNA viruses. Gene transfer vectors derived from adenoviruses (so called adenoviral vectors) have a number of features that make them particularly useful for gene transfer for such purposes. For example, the biology of the adenoviruses is characterized in detail, the adenovirus is not associated with severe human pathology, the virus is extremely efficient in introducing its DNA into the host cell, the virus can infect a wide variety of cells and has a broad host range, the virus can be produced in large quantities with relative ease, and the virus can be rendered replication defective by deletions in the early region 1 (E1) of the viral genome.
The adenovirus (Ad) genome is a linear double-stranded DNA molecule of approximately 36000 base pairs with the 55-kDa terminal protein covalently bound to the 5xe2x80x2 terminus of each strand. The Ad DNA contains identical Inverted Terminal Repeats (ITR) of about 100 base pairs with the exact length depending on the serotype. The viral origins of replication are located within the ITRs exactly at the genome ends. DNA synthesis occurs in two stages. First, the replication proceeds by strand displacement, generating a daughter duplex molecule and a parental displaced strand. The displaced strand is single stranded and can form a so called xe2x80x9cpanhandlexe2x80x9d intermediate, which allows replication initiation and generation of a daughter duplex molecule. Alternatively, replication may proceed from both ends of the genome simultaneously, obviating the requirement to form the panhandle structure. The replication is summarized in FIG. 14 adapted from Lechner et al, (1977) J Mol. Biol. 174:493-510.
During the productive infection cycle, the viral genes are expressed in two phases: the early phase, which is the period up to viral DNA replication, and the late phase, which coincides with the initiation of viral DNA replication. During the early phase only the early gene products, encoded by regions E1, E2, E3 and E4, are expressed, which carry out a number of functions that prepare the cell for synthesis of viral structural proteins (Berk, A. J. (1986) Ann. Rev. Genet. 20: 45-79). During the late phase the late viral gene products are expressed in addition to the early gene products and host cell DNA and protein synthesis are shut off. Consequently, the cell becomes dedicated to the production of viral DNA and of viral structural proteins (Tooze, J. (1981) DNA Tumor Viruses(revised). Cold Spring Harbor Laboratory. Cold Spring Harbor, N.Y.).
The E1 region of adenovirus is the first region of adenovirus expressed after infection of the target cell. This region consists of two transcriptional units, the E1A and E1B genes, both of which are required for oncogenic transformation of primary (embryonal) rodent cultures. The main functions of the E1A gene products are to induce quiescent cells to enter the cell cycle and resume cellular DNA synthesis, and to transcriptionally activate the E1B gene and the other early regions (E2, E3 and E4) of the viral genome. Transfection of primary cells with the E1A gene alone can induce unlimited proliferation (immortalization), but does not result in complete transformation. However, expression of E1A in most cases results in induction of programmed cell death (apoptosis), and only occasionally immortalization is obtained (Jochemsen et al, (1987) EMBO J. 6:3399-3405). Co-expression of the E1B gene is required to prevent induction of apoptosis and for complete morphological transformation to occur. In established immortal cell lines, high level expression of E1A can cause complete transformation in the absence of E1B (Roberts et al, (1985) J Virol. 56:404-413).
The E1 B encoded proteins assist E1A in redirecting the cellular functions to allow viral replication. The E1B 55 kD and E4 33kD proteins, which form a complex that is essentially localized in the nucleus, function in inhibiting the synthesis of host proteins and in facilitating the expression of viral genes. Their main influence is to establish selective transport of viral mRNAs from the nucleus to the cytoplasm, concomittantly with the onset of the late phase of infection. The E1B 21 kD protein is important for correct temporal control of the productive infection cycle, thereby preventing premature death of the host cell before the virus life cycle has been completed. Mutant viruses incapable of expressing the E1B 21 kD gene product exhibit a shortened infection cycle that is accompanied by excessive degradation of host cell chromosomal DNA (deg-phenotype) and in an enhanced cytopathic effect (cyt-phenotype) (Telling et al, (1994) J. Virol 68:541-7). The deg and cyt phenotypes are suppressed when in addition the E1A gene is mutated, indicating that these phenotypes are a function of E1A (White et al, (1988) J Virol. 62: 3445-3454). Furthermore, the E1B 21 kDa protein slows down the rate by which E1A switches on the other viral genes. It is not yet known through which mechanisms E1B 21 kD quenches these E1A dependent functions.
Vectors derived from human adenoviruses, in which at least the E1 region has been deleted and replaced by a gene of interest, have been used extensively for gene therapy experiments in the pre-clinical and clinical phase, and all adenovirus vectors currently used in gene therapy have a deletion in the E1 region, where novel genetic information can be introduced. The E1 deletion renders the recombinant virus replication defective (Stratford-Perricaudet et al, (1991) pp. 51-61. In O. Cohen-Adenaur, and M. Boiron (Eds): Human Gene Transfer, John Libbey Eurotext).
In contrast to, for example, retroviruses, adenoviruses do not integrate into the host cell genome, are able to infect non-dividing cells and are able to efficiently transfer recombinant genes in vivo (Brody et al, (1994) Ann N Y Acad Sci. 716:90-101). These features make adenoviruses attractive candidates for in vivo gene transfer of, for example, suicide or cytokine genes into tumor cells. However, a problem associated with current recombinant adenovirus technology is the possibility of unwanted generation of replication competent adenovirus (RCA) during the production of recombinant adenovirus (Lochmxc3xcller et al, (1994) Hum. Gene Ther. 5:1485-1492; Imler et al, (1996) Gene Ther. 3:75-84). This is caused by homologous recombination between overlapping sequences from the recombinant vector and the adenovirus constructs present in the complementing cell line, such as the 293 cells (Graham et al, (1977) J. Gen. Virol. 36:59-72). RCA in batches to be used in clinical trials is unwanted because RCA i) will replicate in an uncontrolled fashion; ii) can complement replication defective recombinant adenovirus, causing uncontrolled multiplication of the recombinant adenovirus; and iii) batches containing RCA induce significant tissue damage and hence strong pathological side effects (Lochmxc3xciller et al, (1994) Hum. Gene Ther. 5: 1485-1492). Therefore, batches to be used in clinical trials should be proven free of RCA (Ostrove, J. M. (1994) Cancer Gene Ther. 1:125-131).
One of the additional problems associated with the use of recombinant adenovirus vectors is the host defense reaction against treatment with adenovirus. Briefly, recombinant adenoviruses are deleted for the E1 region (see above). The adenovirus E1 products trigger the transcription of the other early genes (E2, E3, E4), which consequently activate expression of the late virus genes. Therefore, it was generally thought that E1 deleted vectors would not express any other adenovirus genes. However, recently it has been demonstrated that some cell types are able to express adenovirus genes in the absence of E1 sequences. This indicates, that some cell types posses the machinery to drive transcription of adenovirus genes. In particular, it was demonstrated that such cells synthesize E2A and late adenovirus proteins. In a gene therapy setting, this means that transfer of the therapeutic recombinant gene to somatic cells not only results in expression of the therapeutic protein but may also result in the synthesis of viral proteins. Cells that express adenoviral proteins are recognized and killed by cytotoxic T Lymphocytes, which both eradicates the transduced cells and causes inflammation (Bout et al, (1994a) Gene Therapy 1:385-394; Engelhardt et al, (1993) Human Gene Therapy 4:759-769; Simon et al, (1993) Human Gene Therapy 4: 771-780). As this adverse reaction hampers gene therapy, several solutions to this problem have been suggested, such as using immunosuppressive gents after treatment, retaining the adenovirus E3 region in the recombinant vector (see patent application EP 9520221B) or using ts mutants of human adenovirus, which have a point mutation in the E2A region (patent WO/28938). However, these strategies to circumvent the immune response have their limitations. The use of ts mutant recombinant adenovirus diminishes the immune response to some extent, but is less effective in preventing pathological responses in the lungs (Engelhardt et al, (1994a) Human Gene Ther. 5:1217-1229). The E2A protein may induce an immune response by itself and it plays a pivotal role in the switch to the synthesis of late adenovirus proteins. Therefore, it is attractive to make recombinant adenoviruses which are mutated in the E2 region, rendering it temperature sensitive (ts), as has been claimed in patent application WO/28938. A major drawback of this system is the fact that, although the E2 protein is unstable at the non-permissive temperature, the immunogenic protein is still synthesized. In addition, it is to be expected that the unstable protein does activate late gene expression, albeit to a low extent. ts125 mutant recombinant adenoviruses have been tested, and prolonged recombinant gene expression was reported (Yang et al, (1994b) Nat Genet. 7:362-369; Engelhardt et al, (1994a) Hum. Gene Ther. 5: 1217-1229; Engelhardt et al, (1994b) Proc Natl Acad Sci USA 91: 6196-200; Yang et al, (1995) J. Virol. 69:2004-2015). However, pathology in the lungs of cotton rats was still high (Engelhardt et al, (1994a) Human Gene Ther. 5:1217-1229), indicating that the use of ts mutants results in only a partial improvement in recombinant adenovirus technology. Others (Fang et al, (1996) Gene Ther. 3:217-222) did not observe prolonged gene expression in mice and dogs using ts125 recombinant adenovirus. An additional difficulty associated with the use of ts125 mutant adenoviruses is that a high frequency of reversion is observed. These revertants are either real revertants or the result of second site mutations (Kruijer et al, (1983) Virology 124:425-433; Nicolas et al, (1981) Virology 108:521-524). Both types of revertants have an E2A protein that functions at normal temperature and therefore have similar toxicity as the wild-type virus.
E1 deleted recombinant adenoviruses are usually made by one of the following method. In the first method, adenovirus DNA, be it wild type (wt) or E1 and/or E3 deleted, is digested with a restriction enzyme e.g. ClaI, to remove the left ITR, packaging signal and at least part of the E1 sequences and the remaining adenovirus genome fragment (1) is purified. Cotransfection of (1) with a linearized adapter construct (2) containing the left ITR, packaging signal, an expression cassette with the gene of interest and adenovirus sequences overlapping with (1) in a cell line complementing for E1 functions (packaging cell line) will give rise to recombinant adenovirus particles by intra-cellular homologous recombination. Alternatively, an adapter construct (3) containing the left ITR, packaging signal, and an expression cassette with the gene of interest is such that it can be ligated to the adenovirus DNA fragment (1) followed by transfection into packaging cells. The disadvantage of these methods is that the purification of (1) is laborious and that incomplete digestion of wt DNA results in introduction of wt adenovirus into the culture leading to contamination. An approach to circumvent this problem has been by the construction of clone pHBG10 described by Bett et al, (1994) Natl. Acad. Sci. USA 91:8802-8806. This plasmid clone contains Ad5 sequences with a deletion of the packaging signal and part of the E1 region and with the viral ITRs attached to each other. However, this clone comprises adenovirus sequences that are also present in E1-complementing cell lines, including those of the present invention (see EP 95201611.1). Furthermore, since the ITRs are attached to each other the clone cannot be linearized, resulting in less efficient recombination with the E1 substitution plasmid.
In the second method, the recombinant adenoviruses is constructed either by homologous recombination in bacteria (Chartier et al, (1996) J. Virol. 70, No. 7:4805-4810; Crouzet et al, (1997) Proc. Natl. Acad. Sci. USA 94:1414-1419) or by cloning into cosmid vectors (Fu et al, (1997) Hum. Gene Ther. 8:1321-1330) and subsequent transfection into an E1 complementing cell line. The disadvantage of this method is that it demands extensive analysis of each generated clone (xcx9c35 kb) by restriction enzyme digestion before transfection to exclude deletions that occurred due to recombination in the bacteria. In addition, the use of cloned adenovirus sequences does not solve the problem of sequence overlap between commonly used packaging cells and recombinant viruses leading to production of RCA during propagation.
A third method that is used is a two-step gene replacement technique in yeast, starting with a complete adenovirus genome (Ad2; Ketner et al, (1994) Proc. Natl. Acad. Sci. USA 91:6186-6190) cloned in a Yeast Artificial Chromosome (YAC) and a plasmid containing adeno sequences to target a specific region in the YAC clone, an expression cassette for the gene of interest and a positive and negative selectable marker. This method requires yeast technology and extensive analysis of each new recombinant clone (even more cumbersome than the above described method, due to the large size of YACs).
A fourth method uses a cosmid clone (pAdexlw; Miyake et al, (1996) Medical Sciences 93:1320-1324) that carries the Ad5 sequence with deletions in the E1 and E3 sequences. This clone has a unique restriction site replacing part of the E1 region that allows insertion of a foreign expression cassette. For the generation of recombinant adenoviruses, a DNA-terminal protein complex (DNA-TPC) is isolated from cells infected with a replication competent adenovirus Ad-dlX (wt Ad5 with an XbaI deletion in the E3 region). This DNA is digested with EcoT22I to remove the 5xe2x80x2 part of the DNA and cotransfected with the cosmid cloned into E1 complementing cells. Intracellular recombination generates the recombinant virus (Miyake et al, (1996) Medical Sciences 93:1320-1324). This method has the disadvantage that replication competent viral DNA is used and that the E1 deletion in the cosmid clone is not enough to remove all overlap with E1 sequences in currently used packaging cell lines including those used in the present invention. Thus, current methods to generate RCA-free recombinant adenoviruses have several disadvantages, including the risk of introducing wild-type viruses in the culture, instability of cloned adenovirus sequences, the necessity to check the complete xcx9c35 kb recombinant clone by restriction analysis for each new virus to be generated, and the system being suitable only for E1 deleted recombinant adenoviruses and much more laborious for use with recombinant adenoviruses comprising E3 substitutions. Furthermore, despite the use of cloned adenovirus DNA in some of the methods, extensive overlap with adenovirus sequences present in commonly used packaging cells like 293 and 911 cells does not solve the problem of appearance of RCA due to homologous recombination during propagation of the virus. Therefore, a need persists for methods and means to produce RCA-free recombinant adenovirus preparations that solve the disadvantages of prior art methods and means discussed above. Gene addition is currently by far the most widely applied gene therapy technique. This is mainly due to the fact that a) homologous recombination is very inefficient and b) for homologous recombination relatively large DNA fragments are required for which no suitable vector systems were available. Thus there is currently an unmet need for vector systems that efficiently introduce large nucleic acid molecules into mammalian cells.
Recombinant adenoviruses are able to efficiently transfer recombinant genes to the rat liver and airway epithelium of rhesus monkeys (Bout et al, (1994b) Human Gene Therapy 5: 3-10; Bout et al, (1994a) Gene Therapy 1: 385-394). In addition, (Vincent et al, (1996) J. Neurosurg 85:648-654; Vincent et al, (1996b) Hum. Gene Ther. 7:197-205) and others (see for example Haddada et al, (1993) Hum Gene Ther 4:703-11) have observed an efficient in vivo adenovirus mediated gene transfer to a variety of tumor cells in vitro and to solid tumors in animal models (lung tumors, glioma) and human xenografts in immunodeficient mice (lung) in vivo (reviewed by Blaese et al, Cancer Gene Ther. 2:291-297).
Generation of minimal adenovirus vectors has been disclosed in WO 94/12649. The method described exploits the function of the protein IX for the packaging of minimal adenovirus vectors (Pseudo Adenoviral Vectors (PAV) in the terminology of WO 94/12649). PAVs are produced by cloning an expression plasmid with the gene of interest between the left-hand (including the sequences required for encapsidation) and the right-hand adenoviral ITRs. The PAV is propagated in the presence of a helper virus. Encapsidation of the PAV is preferred compared to the helper virus because the helper virus is partially defective for packaging (either by virtue of mutations in the packaging signal or by virtue of its size (virus genomes greater than 37.5 kb package inefficiently)). In addition, the authors propose that in the absence of the protein IX gene the PAV will be preferentially packaged. However, neither of these mechanisms appear to be sufficiently restrictive to allow packaging of only PAVs/minimal vectors. The mutations proposed in the packaging signal diminish packaging, but do not provide an absolute block as the same packaging activity is required to propagate the helper virus. Also neither an increase in the size of the helper virus nor the mutation of the protein IX gene will ensure that PAV is packaged exclusively. Thus, the method described in WO 94/12649 is unlikely to be useful for the production of helper-free stocks of minimal adenovirus vectors/PAVs.
Novel compositions and methods are provided for producing recombinant adenoviruses, not only E1-deleted but also minimal adenoviruses which are free of replication competent adenoviruses. The compositions include constructs suitable for the generation of double insert viruses. The system provided by the invention to generate E1 deleted adenoviruses consists of two nucleic acid moieties of which the first is a relatively small and easy to manipulate adapter plasmid containing at least, in an operable configuration, the left ITR, the packaging signal, an expression cassette with the nucleic acid molecule of interest and adenovirus sequences homologous to a part of the second moiety that comprises one, or two or more partially overlapping, nucleic acid molecules that comprise at least all adenovirus sequences necessary for replication and packaging; and the packaging cells of the invention described infra. Co-transfection of the two nucleic acid moieties into the packaging cells leads to homologous recombination between the overlapping sequences in said two nucleic acid moieties. This generates a recombinant viral DNA that is able to replicate and propagate on the packaging cells. The nucleic acid moieties have no sequence overlap with the E1 complementing sequences in packaging cells that can lead to the formation of replication competent adenovirus (RCA). Preferably, at least one of the ITRs on the nucleic acid moieties is flanked by a restriction enzyme recognition site not present in the adenoviral sequences so that the ITR can be made free from vector sequences by digestion of the DNA with that restriction enzyme. In this way initiation of replication occurs more efficiently. The system provided by the present invention also greatly facilitates the production of RCA-free recombinant adenoviruses with further modifications in the adenovirus genome, including but not limited to modifications in the coding regions for the penton base protein or fiber protein or E2A protein.